Title:
Analysis for protein expression, purification, and characterization using E.coli (pGEM-GBR22).
Introduction:
Hundreds of genomes have been successfully sequenced to date, and the data are publicly available, also the advances in large-scale expression and purification of recombinant proteins have paved the way for structural genomics efforts [2]. The stably folded, globular domains of prokaryotic and eukaryotic proteins are a major focus both of the biomedical research community and of our laboratories which are generally suitable for expression in E. coli. Over the years, much effort has been put into optimizing E. coli as an expression host for proteins from higher organisms, which approves that the strategy has generated a wide collection of tools that can be used to increase the yield of soluble protein [1]. Scientists suggest that the protein should be produced as a fusion to an affinity tag because tags dramatically aid in protein purification and rarely adversely affect biological or biochemical activity, however, in selecting which tag to use, one is faced with a daunting number of choices. Despite the lack of a clear winner based on success rate, most of our research groups selected an N-terminal hexahistidin tags that can be removed by site-specific protease [1]. Characterizing the purified protein in some detail reduces the risk of wasting resources on protein material of inadequate quality. It also provides a means to ensure that different batches of the same protein have similar properties [1]. Competent bacterial cells will be transformed with DNA plasmid, grow a starter culture of bacteria, and express the recombinant in order to overexpress a recombinant protein in bacteria. Also the ptoein will not only be purified uing the affinity tag and Ni-NTA resin, but also be analyzed using gel electrophoresis and the smapels prepared during protein purification lab.
Materials & Methods:
For the first day, pGEM-GBR 22 was first expressed in E.Coli was placed in two different agar plates, one with ampicillin and one without ampicillin. Bacteria were left in the incubator and the temperature was set to 37°C. The next day, 0.625mL of bacteria culture was transferred from the tube to the 125mL flasks. The flasks were placed in the shaking incubator and let those grow for 16-24hours at 37°C and 200-350 rpm. Wrap the plates in Para film and save them in the 4°C fridge in VDS section. For the last day for protein expression lab, 500µL sample of culture was collected and dispensed into a labeled eppendorf tube (1.7mL). Using centrifugation method, a pallet was isolated from the cells from the liquid media. After, the pellet, 2.5mL of 1x PBS, and 51µL of lysozyme (50µg/µL) were added to 50mL conical tube. After a week, the pellet was thawed out then lysozyme and cyanase were added to break down the cells. During different stages of the experiment, different samples were gathered to compare the size of protein in the future during protein characterization lab. After, bacteria were centrifuged and the protein is isolated from other substances. Protein was purified using a combination of batch and column chromatography. Resin and buffer was transferred to the top of the column by pouring gently and allow the resin to settle. Using Ni-NTA resin was washed with 5mL of 20mM imidazole in 1xPBS by adding the buffer to the top of the column. The bound protein was eluted by adding 5mL of the buffer containing 250mM imidazole to the top of the column. After, Nanodrop spectrophotometer was used to measure the concentration and the yield of the eluted samples at 280 nm and at the maximal wavelength. Using six samples collected from protein purification lab, pre-cast gel (4-20%), mini-PROTEAM tank with 500mL 1x TGS, gel was run for 25 minutes at 200V. The gel was stained Imperial Protein Stain for 1.5 hours, and after, nanopure water was added to the gel to rinse out the gel twice. Finally, the gel was covered in Waltman paper and dried.
Results:
Figure 1. Agar plate with E.coli - BL21(DE3), DNA pGEM-gbr22 and Ampicillin. VDS. Day 2 (Morning). 03/07/2013.
Figure 2. Agar plate with E.coli - BL21(DE3), No DNA control and Ampicillin. VDS, Day 2 (Morning). 03/07/2013
Figure 3. Agar plate with random bacterial cells swab from the cell phone screen. VDS. Day 2 (Morning). 03/07/2013.
Figure 4. Large bacterial cell culture with E.coli - BL21(DE3), DNA plasmid - pGEM-gbr22 and Media - LB + AMP. VDS. Day 2 (Evening). 03/08/2013.
Figure 5. E. coli - BL21(DE3), DNA plasmid - (pGEM-gbr22) and Media (LB+AMP). VDS. Day3. 03/08/2013. Wet pellet of 0.62g centrifuged bacterial cells in purple.
Figure 6. Elusion buffer 1
Figure 7. Elution buffer 2
Graph 1. Reading 1 for elution 1 measuring at 280nm
Graph 2. Reading 2 for elution 2 measuring at 280nm.
Graph 3. Reading 1 for elution 2 measuring at 280nm.
Graph 5. Reading 1 for elution 1 measuring at the maximal wavelength
Graph 6. Reading 2 for elution 1 measuring at the maximal wavelength
Graphv 7. Reading 1 for elutiokn 2 measuring at the maximal wavelength.
Graph 8. Reading 2 for elution 2 measuring at the maximal wavelength.
Figure 8. Rinsed SDS gel after electrophoresis at 200V for 25 minutes. The first lane is the PageRuler pre-stained protein ladder. Second lane is sample one. Third lane is empty due to the absence of sampel two, and the subsequent lanes are samples 3,4,5 and 6. Obtained with BIO-RAD precast gel and a mini- PROTEIN electrophoresis tank.
Figure 9. SDS-PAGE profile of the Thermo Sientific PageRuler Prestained protein ladder product #26616. Information was obtained from PageRule website.
Figure 10. Dried SDS-gel after using drying bed at 75°C for 1.5 hours. First lane is the PageRuler prestained protein ladder. Second lane is sample one. Third lane is empty dur to the absence of sample two, and the subsequent lanes are sample 3,4,5 and 6.
E.coli (PB21DE3) was used due to the fact the strain that was used for this lab is not harmful. Plasmid is negative charged, and bacterial cells also have negative charge. On the other hand, outside of cells have positive charge due to the calcium ions, which make overall neutral. Plasmid was added into bacterial cells and coded as purple to distinguish those specific bacterial cells from other bacteria. The reason why bacterial cells were successfully transformed on agar plate with DNA plasmid is because plasmid is resistant to Ampicillin. During the process of making agar plate for this experiment, ampicillin was added into the plate to prevent other bacteria to grow. Since only bacteria with DNA have an ability to survive and grow, plate with DNA appears pink/purple after transformation. SOC and LB media were used to make more bacterial cells to survive, since those are nutrient rich media. Also, putting into ice make cells to prevent moving, and putting plasmid first into ice bucket is important because plasmid is sensitive to heat.
During protein purification, E.coli cells express purple protein was lysed to obtain protein. Also, only protein from bacterial cells was collected after centrifugation which divided cells into two different layers (DNA part and protein). Afterwards, Protein was purified using Ni-NTA first, since Nickel is attached to bead resin complex which is the reason why protein cannot go through, also Histidine tags of protein sticks to nickel, therefore retaining the protein. However, when imidazole was added Ni-NTA binds to imidazole instead of protein, which made proteins to come out. However, when imidazole was added Ni-NTA binds to imidazole instead of protein, which made proteins to come out with buffer. Elution 1 contains most of the protein since the method stated above was used; therefore, this explains the reason why elution 2 appears as clear while elution 1 appears as purple.
Finally, the main purpose of this lab was estimating the purity of the protein sample obtained from protein purification lab Different samples was collected after different steps. Sample 5 was used in this lab since sample 5 was the most purified sample. One can assume that sample 5 was well purified due to the fact that the gel had only one clear band, which confirms the purity of this protein is close to 100 percent. Also, it is certain that the protein band from sample #5 is in fact gbr22, since protein ladder confirms that the band is located approximately ~25kDa.
Possible error that one can have during the first experiment is contamination. Since bacterial cells are easy to get contaminated, proper sterilization technique is required while handing any substances putting into bacteria. However, since other bacteria from lab station and hands could possibly contaminate test tubes and agar plates, it is certain that bacterial cell culture might have some errors. Also, measurement error can also greatly affect the result. Since many different substances such as LB media, PBS stock, and etc. were added using pipettes, it can result inaccurate amount of substance were added. These errors can be prevented by using micro-pipettors instead of regular pipettes and work near the flam and minimize the time when transporting substance into the test tube. Positive control for protein expression was plate DNA, and negative control was plate with no DNA.
During protein purification, one could possibly measure incorrect amount of solutions when they create buffer solutions, which might leads to incorrect reading during spectrophotometer measurement. Also, one needs to keep solutions cold to prevent enzyme degradation. However, frequent temperature change on enzyme can also possibly result having inaccurate buffer solutions.
In protein characterization, estimating purity of gel can be calculated with an error, since one could see several lightly colored bands besides one vey dark band. Moreover, gel electrophoresis could also have one a wrong result if the voltage was not set to 200V.
Conclusions:
Bacterial cells were used for this experiment, since it is easy to express compare to human cells. One could see cultured bacteria were appeared as purple/pink which indicates the plasmid was successfully transformed into bacterial cells. This lab/experiment can be used in VDS lab or any biology-related labs when one artificially transform certain DNA into the cells or produce copies of DNA. It is also useful to identify certain gene by inserting code for specific color into its gene.
During protein purification, Ni-NTA affinity syringe filer, centrifugation and lysate were used to purify protein from E.coli cells. Also, the concentration of protein was calculated using the different wavelength and spectrophotometer. In the future, purified protein will be used to characterize protein using gel-electrophoresis and UV-VIS spectroscopy.
Since protein purification and gel electrophoresis gave a result that the purity of the protein gbr-22 was close to 100 percent. In VDS, other protein samples will need to be characterized and purified protein characterization is important to confirm the identity of a potential drug target and determine the purity of the sample.
References: [1] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods2008,5 (2), 135-146. [2] Vedadi, M.; Arrowsmith, C. H.; Allali-Hassani, A.; Senisterra, G.; Wasney, G. A.; Biophysical characterization of recombinant proteins: A key to higher structural genomics success. J Struct Biol. 2010, 172 (1-2): 107-119.
Analysis for protein expression, purification, and characterization using E.coli (pGEM-GBR22).
Introduction:
Hundreds of genomes have been successfully sequenced to date, and the data are publicly available, also the advances in large-scale expression and purification of recombinant proteins have paved the way for structural genomics efforts [2]. The stably folded, globular domains of prokaryotic and eukaryotic proteins are a major focus both of the biomedical research community and of our laboratories which are generally suitable for expression in E. coli. Over the years, much effort has been put into optimizing E. coli as an expression host for proteins from higher organisms, which approves that the strategy has generated a wide collection of tools that can be used to increase the yield of soluble protein [1]. Scientists suggest that the protein should be produced as a fusion to an affinity tag because tags dramatically aid in protein purification and rarely adversely affect biological or biochemical activity, however, in selecting which tag to use, one is faced with a daunting number of choices. Despite the lack of a clear winner based on success rate, most of our research groups selected an N-terminal hexahistidin tags that can be removed by site-specific protease [1]. Characterizing the purified protein in some detail reduces the risk of wasting resources on protein material of inadequate quality. It also provides a means to ensure that different batches of the same protein have similar properties [1]. Competent bacterial cells will be transformed with DNA plasmid, grow a starter culture of bacteria, and express the recombinant in order to overexpress a recombinant protein in bacteria. Also the ptoein will not only be purified uing the affinity tag and Ni-NTA resin, but also be analyzed using gel electrophoresis and the smapels prepared during protein purification lab.
Materials & Methods:
For the first day, pGEM-GBR 22 was first expressed in E.Coli was placed in two different agar plates, one with ampicillin and one without ampicillin. Bacteria were left in the incubator and the temperature was set to 37°C. The next day, 0.625mL of bacteria culture was transferred from the tube to the 125mL flasks. The flasks were placed in the shaking incubator and let those grow for 16-24hours at 37°C and 200-350 rpm. Wrap the plates in Para film and save them in the 4°C fridge in VDS section. For the last day for protein expression lab, 500µL sample of culture was collected and dispensed into a labeled eppendorf tube (1.7mL). Using centrifugation method, a pallet was isolated from the cells from the liquid media. After, the pellet, 2.5mL of 1x PBS, and 51µL of lysozyme (50µg/µL) were added to 50mL conical tube. After a week, the pellet was thawed out then lysozyme and cyanase were added to break down the cells. During different stages of the experiment, different samples were gathered to compare the size of protein in the future during protein characterization lab. After, bacteria were centrifuged and the protein is isolated from other substances. Protein was purified using a combination of batch and column chromatography. Resin and buffer was transferred to the top of the column by pouring gently and allow the resin to settle. Using Ni-NTA resin was washed with 5mL of 20mM imidazole in 1xPBS by adding the buffer to the top of the column. The bound protein was eluted by adding 5mL of the buffer containing 250mM imidazole to the top of the column. After, Nanodrop spectrophotometer was used to measure the concentration and the yield of the eluted samples at 280 nm and at the maximal wavelength. Using six samples collected from protein purification lab, pre-cast gel (4-20%), mini-PROTEAM tank with 500mL 1x TGS, gel was run for 25 minutes at 200V. The gel was stained Imperial Protein Stain for 1.5 hours, and after, nanopure water was added to the gel to rinse out the gel twice. Finally, the gel was covered in Waltman paper and dried.
Results:
Beer's Law Calculation:
A=Ɛbc
A= absorbance
Ɛ= Molar absorptivity (avg. Abs/concentration mass)
c= concentration (mol/L)
b= path length (1mm)
Molecular weight: 25794.2 g/mol
0.410 = (38850)(1mm)( c )
( c ) = = 0.0000105534 mols/L
0.0000105534 mols/L * 25794.2 g/mol = 0.27221651 mg/mL @ 280nm
Discussion:
E.coli (PB21DE3) was used due to the fact the strain that was used for this lab is not harmful. Plasmid is negative charged, and bacterial cells also have negative charge. On the other hand, outside of cells have positive charge due to the calcium ions, which make overall neutral. Plasmid was added into bacterial cells and coded as purple to distinguish those specific bacterial cells from other bacteria. The reason why bacterial cells were successfully transformed on agar plate with DNA plasmid is because plasmid is resistant to Ampicillin. During the process of making agar plate for this experiment, ampicillin was added into the plate to prevent other bacteria to grow. Since only bacteria with DNA have an ability to survive and grow, plate with DNA appears pink/purple after transformation. SOC and LB media were used to make more bacterial cells to survive, since those are nutrient rich media. Also, putting into ice make cells to prevent moving, and putting plasmid first into ice bucket is important because plasmid is sensitive to heat.
During protein purification, E.coli cells express purple protein was lysed to obtain protein. Also, only protein from bacterial cells was collected after centrifugation which divided cells into two different layers (DNA part and protein). Afterwards, Protein was purified using Ni-NTA first, since Nickel is attached to bead resin complex which is the reason why protein cannot go through, also Histidine tags of protein sticks to nickel, therefore retaining the protein. However, when imidazole was added Ni-NTA binds to imidazole instead of protein, which made proteins to come out. However, when imidazole was added Ni-NTA binds to imidazole instead of protein, which made proteins to come out with buffer. Elution 1 contains most of the protein since the method stated above was used; therefore, this explains the reason why elution 2 appears as clear while elution 1 appears as purple.
Finally, the main purpose of this lab was estimating the purity of the protein sample obtained from protein purification lab Different samples was collected after different steps. Sample 5 was used in this lab since sample 5 was the most purified sample. One can assume that sample 5 was well purified due to the fact that the gel had only one clear band, which confirms the purity of this protein is close to 100 percent. Also, it is certain that the protein band from sample #5 is in fact gbr22, since protein ladder confirms that the band is located approximately ~25kDa.
Possible error that one can have during the first experiment is contamination. Since bacterial cells are easy to get contaminated, proper sterilization technique is required while handing any substances putting into bacteria. However, since other bacteria from lab station and hands could possibly contaminate test tubes and agar plates, it is certain that bacterial cell culture might have some errors. Also, measurement error can also greatly affect the result. Since many different substances such as LB media, PBS stock, and etc. were added using pipettes, it can result inaccurate amount of substance were added. These errors can be prevented by using micro-pipettors instead of regular pipettes and work near the flam and minimize the time when transporting substance into the test tube. Positive control for protein expression was plate DNA, and negative control was plate with no DNA.
During protein purification, one could possibly measure incorrect amount of solutions when they create buffer solutions, which might leads to incorrect reading during spectrophotometer measurement. Also, one needs to keep solutions cold to prevent enzyme degradation. However, frequent temperature change on enzyme can also possibly result having inaccurate buffer solutions.
In protein characterization, estimating purity of gel can be calculated with an error, since one could see several lightly colored bands besides one vey dark band. Moreover, gel electrophoresis could also have one a wrong result if the voltage was not set to 200V.
Conclusions:
Bacterial cells were used for this experiment, since it is easy to express compare to human cells. One could see cultured bacteria were appeared as purple/pink which indicates the plasmid was successfully transformed into bacterial cells. This lab/experiment can be used in VDS lab or any biology-related labs when one artificially transform certain DNA into the cells or produce copies of DNA. It is also useful to identify certain gene by inserting code for specific color into its gene.
During protein purification, Ni-NTA affinity syringe filer, centrifugation and lysate were used to purify protein from E.coli cells. Also, the concentration of protein was calculated using the different wavelength and spectrophotometer. In the future, purified protein will be used to characterize protein using gel-electrophoresis and UV-VIS spectroscopy.
Since protein purification and gel electrophoresis gave a result that the purity of the protein gbr-22 was close to 100 percent. In VDS, other protein samples will need to be characterized and purified protein characterization is important to confirm the identity of a potential drug target and determine the purity of the sample.
References:
[1]
Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-146.
[2]
Vedadi, M.; Arrowsmith, C. H.; Allali-Hassani, A.; Senisterra, G.; Wasney, G. A.; Biophysical characterization of recombinant proteins: A key to higher structural genomics success. J Struct Biol. 2010, 172 (1-2): 107-119.